Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti

Abstract

Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterward IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg(2+) or Mn(2+) but not Zn(2+) for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.

Figure 1. IPPI assay outline

Isomerization of IPP to DMAPP is catalyzed by IPPI. During the reaction an electrophilic attack by a proton from water occurs on the IPP double bond to yield the carbocation intermediate, afterwards the C-2 pro-R hydrogen of the intermediate is stereospecifically eliminated. DMAPP is converted to isoprene by acidic hydrolysis and dehydration using phosphoric acid. Volatile isoprene is adsorbed using a SPME fiber and detected by gas chromatography.

Figure 2. AaIPPI amino acid sequence

Predicted α-helices and β-sheets are labeled on top and shown inside a box. IPPI conserved sequence motifs are highlighted in bold and underlined. (▲): Residues important for metal coordination. (*): Residues important for catalysis. (#): Residues important for interaction with the diphosphate moiety of the substrate. The putative Nudix motif is highlighted by a grey box.

Figure 3. Effect of metal cofactors

Different concentrations of Mg²⁺ (A) or Mn²⁺ (B) were added to samples and IPPI activity was analyzed. Activities are expressed as increases relative to the activity of the control without metal cofactor (fold increase). Each data point is the mean ± S.E.M of 2–4 independent replicates.

Figure 4. Iodoacetamide inhibition of AaIPPI

Percentages of inhibition increased as increasing concentrations of iodoacetamide were added to the sample up to 500 μM. Controls had no inhibitors. Each data point is the mean ± S.E.M of 2 independent replicates.

Figure 5. Saturation kinetics of AaIPPI

Plot of initial velocity of AaIPPI activity versus IPP concentration. Each data point is the mean ± S.E.M of 3 independent replicates.

Figure 6. Tissue specific expression of AaIPPI mRNA

All female tissues were dissected from three-day old sugar-fed females. CA: corpora allata-corpora cardiaca; OV: ovary; HG: hindgut; BR: brain; MT: Malpighian tubules; HT: heart; MG: midgut and FB: fat body. The insert shows the male testis (TS) and accessory glands (AG). AaIPPI mRNAs are expressed as copy number/10,000 copies of rpL32 mRNA. Each RT-PCR data point is mean ± S.E.M of two independent biological replicates of 10 tissue samples. These studies are an expansion of studies published by Nouzova et al. (2011).

Figure 7. Developmental expression of AaIPPI mRNA in the CA

Expression of AaIPPI mRNA in CA-CC of pupa, sugar-fed and blood-fed adult female. AaIPPI mRNA is expressed as copy number of AaIPPI mRNA/10,000 copies of rpL32 mRNA. Each RT-PCR data point is mean ± S.E.M of two independent biological replicates of 20 CA-CC. JH biosynthesis values are based on Li et al. (2003) and are expressed as fmol/CA/h. These studies are an expansion of studies published by Nouzova et al. (2011).